This project will employ a novel approach to the empirical identification of candidate genes influencing total antioxidant status, an index of oxidative stress that is related to cardiovascular disease risk. Using existing quantitative transcriptional profiles obtained from lymphocyte samples from 1,240 Mexican American individuals in the San Antonio Family Heart Study, we have identified approximately 1,400 transcripts that exhibit highly significant evidence for cis-regulation as inferred from quantitative linkage analysis. Each of these cis-regulated transcripts has been examined for association with TAS levels. Using this large-scale genome-wide expression data, we have discovered approximately 150 cis-regulated genes that also are significantly correlated with TAS levels. The proposed project will exploit this genome-wide expression-based information to rapidly identify regulatory sequence variants that influence transcriptional levels of these novel empirically-chosen candidate genes and to assess their influence on human plasma total antioxidant status. In order to identify genes involved in oxidative stress that are relevant to cardiovascular disease risk, we will examine 75 novel empirically-chosen candidates. Our prior evidence for cis-acting sequence variation can be exploited as a probabilistic causal anchor to maximize our chance for finding functional variation within the proximal promoters of the chosen genes. For each gene, we will (1) resequence approximately two kilobases of putative promoter region in 182 founder individuals to identify promoter variants; (2) genotype all detected promoter variation in the 1,240 SAFHS samples for which we have transcriptional profiles; (3) test whether promoter sequence variants are associated with gene expression levels of the appropriate candidate gene; (4) test for associations between promoter sequence variants and plasma total antioxidant levels; (5) confirm observed associations with total antioxidant levels in an independent sample of Mexican American families; and (6) perform preliminary functional analyses of promoter variants influencing TAS levels. The proposed research should increase the pace of discovery of novel genes underlying human variation in accommodation of oxidative stress in relation to CVD risk. By focusing on genes whose transcripts show evidence for both cis-regulatory variation and a strong relationship with antioxidant status, we should maximize our probability for finding causal genetic variants influencing CVD risk. PUBLIC HEALTH RELEVANCE: Atherosclerosis is the leading cause of mortality in North America and is predicted to attain a similar distinction on a global scale within the next twenty years. In this project, we will employ a novel strategy that should increase the pace of discovery of genes that are involved in heart disease risk. Such knowledge of some of the genes involved in susceptibility to this disease will contribute to our understanding of the causes of atherosclerosis and potentially speed the search for new treatments.